Recent Advances in Single-Cell Profiling and Multispecific Therapeutics: Paving the Way for a New Era of Precision Medicine Targeting Cardiac Fibroblasts

Abstract Purpose of Review Cardiac fibroblast activation contributes to fibrosis, maladaptive remodeling and heart failure progression. This review summarizes the latest findings on cardiac fibroblast activation dynamics derived from single-cell transcriptomic analyses and discusses how this information may aid the development of new multispecific medicines. Recent Findings Advances in single-cell gene expression technologies have led to the discovery of distinct fibroblast subsets, some of which are more prevalent in diseased tissue and exhibit temporal changes in response to injury. In parallel to the rapid development of single-cell platforms, the advent of multispecific therapeutics is beginning to transform the biopharmaceutical landscape, paving the way for the selective targeting of diseased fibroblast subpopulations. Summary Insights gained from single-cell technologies reveal critical cardiac fibroblast subsets that play a pathogenic role in the progression of heart failure. Combined with the development of multispecific therapeutic agents that have enabled access to previously “undruggable” targets, we are entering a new era of precision medicine.

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A Transcriptional Switch Governing Fibroblast Plasticity Underlies Reversibility of Chronic Heart Disease

10.1101/2020.07.21.214874 ◽

2020 ◽

Author(s):

Michael Alexanian ◽

Pawel F. Przytycki ◽

Rudi Micheletti ◽

Arun Padmanabhan ◽

Lin Ye ◽

...

Keyword(s):

Heart Disease ◽

Cardiac Function ◽

Single Cell ◽

Cardiac Dysfunction ◽

Cardiac Fibroblasts ◽

Regulatory Elements ◽

Heart Tissue ◽

Chromatin Accessibility ◽

Fibroblast Activation ◽

Transcriptional Switch

AbstractIn diseased organs, stress-activated signaling cascades alter chromatin, triggering broad shifts in transcription and cell state that exacerbate pathology. Fibroblast activation is a common stress response that worsens lung, liver, kidney and heart disease, yet its mechanistic basis remains poorly understood1,2. Pharmacologic inhibition of the BET family of transcriptional coactivators alleviates cardiac dysfunction and associated fibrosis, providing a tool to mechanistically interrogate maladaptive fibroblast states and modulate their plasticity as a potential therapeutic approach3–8. Here, we leverage dynamic single cell transcriptomic and epigenomic interrogation of heart tissue with and without BET inhibition to reveal a reversible transcriptional switch underlying stress-induced fibroblast activation. Transcriptomes of resident cardiac fibroblasts demonstrated robust and rapid toggling between the quiescent fibroblast and activated myofibroblast state in a manner that directly correlated with BET inhibitor exposure and cardiac function. Correlation of single cell chromatin accessibility with cardiac function revealed a novel set of reversibly accessible DNA elements that correlated with disease severity. Among the most dynamic elements was an enhancer regulating the transcription factor MEOX1, which was specifically expressed in activated myofibroblasts, occupied putative regulatory elements of a broad fibrotic gene program, and was required for TGFβ-induced myofibroblast activation. CRISPR interference of the most dynamic cis-element within the enhancer, marked by nascent transcription, prevented TGFβ-induced activation of Meox1. These findings identify MEOX1 as a central regulator of stress-induced myofibroblast activation associated with cardiac dysfunction. The plasticity and specificity of the BET-dependent regulation of MEOX1 in endogenous tissue fibroblasts provides new trans- and cis- targets for treating fibrotic disease.

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Soluble St2 Induces Cardiac Fibroblast Activation and Collagen Synthesis via Neuropilin-1

Cells ◽

10.3390/cells9071667 ◽

2020 ◽

Vol 9 (7) ◽

pp. 1667 ◽

Cited By ~ 1

Author(s):

Lara Matilla ◽

Vanessa Arrieta ◽

Eva Jover ◽

Amaia Garcia-Peña ◽

Ernesto Martinez-Martinez ◽

...

Keyword(s):

Collagen Synthesis ◽

Cardiac Fibrosis ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Interleukin 1 ◽

Cardiac Fibroblast ◽

Pathogenic Role ◽

Fibroblast Activation ◽

Neuropilin 1 ◽

Human Cardiac Fibroblasts

Circulating levels of soluble interleukin 1 receptor-like 1 (sST2) are increased in heart failure and associated with poor outcome, likely because of the activation of inflammation and fibrosis. We investigated the pathogenic role of sST2 as an inductor of cardiac fibroblasts activation and collagen synthesis. The effects of sST2 on human cardiac fibroblasts was assessed using proteomics and immunodetection approaches to evidence the upregulation of neuropilin-1 (NRP-1), a regulator of the profibrotic transforming growth factor (TGF)-β1. In parallel, sST2 increased fibroblast activation, collagen and fibrosis mediators. Pharmacological inhibition of nuclear factor-kappa B (NF-κB) restored NRP-1 levels and blocked profibrotic effects induced by sST2. In NRP-1 knockdown cells, sST2 failed to induce fibroblast activation and collagen synthesis. Exogenous NRP-1 enhanced cardiac fibroblast activation and collagen synthesis via NF-κB. In a pressure overload rat model, sST2 was elevated in association with cardiac fibrosis and was positively correlated with NRP-1 expression. Our study shows that sST2 induces human cardiac fibroblasts activation, as well as the synthesis of collagen and profibrotic molecules. These effects are mediated by NRP-1. The blockade of NF-κB restored NRP-1 expression, improving the profibrotic status induced by sST2. These results show a new pathogenic role for sST2 and its mediator, NRP-1, as cardiac fibroblast activators contributing to cardiac fibrosis.

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Cardiac Fibroblast Diversity

Annual Review of Physiology ◽

10.1146/annurev-physiol-021119-034527 ◽

2020 ◽

Vol 82 (1) ◽

pp. 63-78 ◽

Cited By ~ 8

Author(s):

Michelle D. Tallquist

Keyword(s):

Extracellular Matrix ◽

Single Cell ◽

Future Development ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Pathological Condition ◽

Cardiac Fibroblast ◽

Lineage Tracing ◽

Single Cell Sequencing ◽

Disease States

Cardiac fibrosis is a pathological condition that occurs after injury and during aging. Currently, there are limited means to effectively reduce or reverse fibrosis. Key to identifying methods for curbing excess deposition of extracellular matrix is a better understanding of the cardiac fibroblast, the cell responsible for collagen production. In recent years, the diversity and functions of these enigmatic cells have been gradually revealed. In this review, I outline current approaches for identifying and classifying cardiac fibroblasts. An emphasis is placed on new insights into the heterogeneity of these cells as determined by lineage tracing and single-cell sequencing in development, adult, and disease states. These recent advances in our understanding of the fibroblast provide a platform for future development of novel therapeutics to combat cardiac fibrosis.

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Increased release of serotonin from rat primary isolated adult cardiac myofibroblasts

Scientific Reports ◽

10.1038/s41598-021-99632-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Emiri Tarbit ◽

Indu Singh ◽

Jason Nigel Peart ◽

Svetlana Bivol ◽

Roselyn Barbara Rose’Meyer

Keyword(s):

Heart Failure ◽

Protein Expression ◽

Western Blot ◽

Serotonin Receptors ◽

Serotonin Receptor ◽

Cardiac Fibroblasts ◽

Cardiac Fibroblast ◽

Receptor Protein ◽

Receptor System ◽

Cardiac Myofibroblasts

AbstractElevated blood serotonin levels have been observed in patients with heart failure and serotonin has a role in pathological cardiac function. The serotonin receptor system was examined in adult rat isolated cardiac fibroblast and myofibroblast cells. This is one of the first studies that has investigated serotonin receptors and other proteins involved in the serotonin receptor system in rat cardiac fibroblast and myofibroblast cells. Rat primary cardiac fibroblasts were isolated and transformed into myofibroblasts using 5 ng/ml TGF-β1. Transformation of cells to myofibroblasts was confirmed with the presence of α-smooth muscle actin using Western blot. Serotonin metabolism and receptor protein expression was assessed using Western blot techniques and serotonin levels measured using ELISA. The 5-HT1A, 5-HT2A and 5-HT2B receptors were found to be present in both rat cardiac fibroblasts and myofibroblast cells, however no significance in protein expression between the two cell types was found (P > 0.05). In this study a significant increase in the serotonin transporter (SERT), tryptophan hydroxylase 1 and extracellular serotonin levels was observed in rat cardiac myofibroblasts when compared to fibroblasts (P < 0.05). These results suggest that serotonin levels may rise in parallel with cardiac myofibroblast populations and contribute to the pathogenesis of heart failure via serotonin receptors.

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From single gene analysis to single cell profiling: a new era for precision medicine

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-020-01549-3 ◽

2020 ◽

Vol 39 (1) ◽

Cited By ~ 1

Author(s):

Maria Teresa Di Martino ◽

Stefania Meschini ◽

Katia Scotlandi ◽

Chiara Riganti ◽

Enrico De Smaele ◽

...

Keyword(s):

Precision Medicine ◽

Single Cell ◽

Single Gene ◽

Gene Analysis ◽

New Era ◽

Single Cell Profiling

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Abstract 318: Identification of Novel Cardiac Fibroblast Enriched Genes

Circulation Research ◽

10.1161/res.115.suppl_1.318 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

Jessica M Swonger ◽

Michelle D Tallquist

Keyword(s):

Differentially Expressed Genes ◽

Heart Function ◽

Cardiac Fibroblasts ◽

Cardiac Injury ◽

The United States ◽

Cardiac Fibroblast ◽

Differentially Expressed ◽

Fibroblast Activation ◽

Helix Loop Helix ◽

Future Work

The leading cause of death in the United States is heart disease. While current therapies have reduced mortality, patients surviving the initial stages of cardiac injury are left with long-term disruption of heart function including fibrosis. Pathological fibrosis in the heart is caused by excess proliferation and deposition of extracellular matrix primarily by cardiac fibroblasts (CFs). One factor required for cardiac fibroblast formation is the basic helix-loop-helix transcription factor, TCF21 (epicardin/Pod1/capsulin). Previous studies from our lab have shown that Tcf21 null embryos lack CFs. Our current work focuses on identifying genes downstream of TCF21. Deep-sequencing identified over one hundred differentially expressed genes when comparing embryonic hearts from Tcf21 nulls to wild types. We have verified a subset of these differentially expressed genes by qPCR and have demonstrated that these genes are also expressed in cultured primary cardiac fibroblasts. Future work will focus on determining the function of these genes during fibroblast activation and determine which of these genes are directly regulated by TCF21. The elucidation of cardiac fibroblast specific genes and their function will provide much needed information for identification of therapeutic targets aimed at cardiac fibroblast activation.

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The Cardiac Fibroblast in heart failure – an inflammatory cell

European Heart Journal ◽

10.1093/ehjci/ehaa946.3731 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

T Thottakara ◽

V.M Dhople ◽

S Voss ◽

J Schoen ◽

U Voelker ◽

...

Keyword(s):

Gene Expression ◽

Heart Failure ◽

Expression Analysis ◽

Cardiac Fibroblasts ◽

Cardiac Fibroblast ◽

Cell Culture Supernatant ◽

Type I ◽

Cardiac Inflammation ◽

Ecm Proteins ◽

Tnf Α

Abstract Introduction Cardiac inflammation with subsequent remodeling of the extracellular matrix (ECM) with fibrosis formation is an important precursor of heart failure. In myocardial inflammation release of cytokines, such as TNF-α was previously linked to the development of fibrosis. However, their exact role in fibroblasts activation, leading to their transformation into myofibroblasts and ultimately fibrosis is unknown. Therefore, the purpose of this work was to study the secretome of isolated cardiac fibroblasts of heart failure patients in vitro. Methods and results Human cardiac fibroblasts were obtained from endomyocardial biopsies of patients with reduced ejection fraction. Secretome- and gene expression analysis was performed on TNF-α (10 ng/ml) and TGF-β (5 ng/ml) treated and untreated fibroblasts. The analysis of the secretome in cell culture supernatant of these fibroblasts was performed by nano-LC-ESI tandem mass spectrometry. Subsequently, RNA was isolated from fibroblasts and expression analysis was performed using affymetrix gene chips and verified for the stimulation experiments using TaqMan. The secretome and gene expression studies are constistent regarding the most frequent ECM components. The proteins involved in the construction of the ECM accounted for 56% of the total protein intensity. The fibronectin represented the largest proportion of ECM proteins (50%), with collagens accounting for 29% and MMPs and TIMPs for approximately 5% and a small proportion of cytokines. In the secretome as well as in the gene expression, Il-6 and Il-8 showed the highest proportion of cytokines and type I as well as type IV (each approx. 40%) of collagens. MMP2, MMP1 and TIMP1 accounted for the largest proportion of MMPs and TIMPs. Stimulation with TGF-β and TNF-α led to changes in the composition of the cytokines and ECM proteins. After the stimulation with TNF-α, the proportion of cytokines increased from 3.1% to 6.6%, while the proportion of ECM proteins decreased slightly. Accordingly, after stimulation with TGF-β a well-known inductor of fibrosis, the proportion of collagens and other matrix proteins increased, while the proportion of cytokines decreased slightly. Conclusion In this study, we identified the most abundant proteins of the ECM and demonstrated their expansion caused by TGF-β stimulation of fibroblasts. Surprisingly, TNF-α led to an increase of cytokines secreted by the fibroblasts with a minimal reduction of ECM proteins. Here, the differentiated regulation of the fibroblast in the transformation of the ECM could be seen. TNF-α stimulation demonstrated to increase gene expression of the cytokines in fibroblasts, which in turn maintain inflammation with chemotactic effect. Hence, cardiac fibroblasts seem to be supporting cells for cardiac inflammation. There seemed to be no pro-fibrotic effect. In this work we could show the pro-inflammatory role of the cardiac fibroblast upon inflammatory stimulation. Funding Acknowledgement Type of funding source: None

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SKI activates the Hippo pathway via LIMD1 to inhibit cardiac fibroblast activation

Basic Research in Cardiology ◽

10.1007/s00395-021-00865-9 ◽

2021 ◽

Vol 116 (1) ◽

Author(s):

Natalie M. Landry ◽

Sunil G. Rattan ◽

Krista L. Filomeno ◽

Thomas W. Meier ◽

Simon C. Meier ◽

...

Keyword(s):

Cardiac Fibroblasts ◽

Cardiac Fibroblast ◽

Negative Regulator ◽

Expression Vectors ◽

Cell Phenotype ◽

Hippo Signaling ◽

Fibroblast Activation ◽

Elastic Substrates ◽

Human Cardiac Fibroblasts

AbstractWe have previously shown that overexpression of SKI, an endogenous TGF-β1 repressor, deactivates the pro-fibrotic myofibroblast phenotype in the heart. We now show that SKI also functions independently of SMAD/TGF-β signaling, by activating the Hippo tumor-suppressor pathway and inhibiting the Transcriptional co-Activator with PDZ-binding motif (TAZ or WWTR1). The mechanism(s) by which SKI targets TAZ to inhibit cardiac fibroblast activation and fibrogenesis remain undefined. A rat model of post-myocardial infarction was used to examine the expression of TAZ during acute fibrogenesis and chronic heart failure. Results were then corroborated with primary rat cardiac fibroblast cell culture performed both on plastic and on inert elastic substrates, along with the use of siRNA and adenoviral expression vectors for active forms of SKI, YAP, and TAZ. Gene expression was examined by qPCR and luciferase assays, while protein expression was examined by immunoblotting and fluorescence microscopy. Cell phenotype was further assessed by functional assays. Finally, to elucidate SKI’s effects on Hippo signaling, the SKI and TAZ interactomes were captured in human cardiac fibroblasts using BioID2 and mass spectrometry. Potential interactors were investigated in vitro to reveal novel mechanisms of action for SKI. In vitro assays on elastic substrates revealed the ability of TAZ to overcome environmental stimuli and induce the activation of hypersynthetic cardiac myofibroblasts. Further cell-based assays demonstrated that SKI causes specific proteasomal degradation of TAZ, but not YAP, and shifts actin cytoskeleton dynamics to inhibit myofibroblast activation. These findings were supported by identifying the bi-phasic expression of TAZ in vivo during post-MI remodeling and fibrosis. BioID2-based interactomics in human cardiac fibroblasts suggest that SKI interacts with actin-modifying proteins and with LIM Domain-containing protein 1 (LIMD1), a negative regulator of Hippo signaling. Furthermore, we found that LATS2 interacts with TAZ, whereas LATS1 does not, and that LATS2 knockdown prevented TAZ downregulation with SKI overexpression. Our findings indicate that SKI’s capacity to regulate cardiac fibroblast activation is mediated, in part, by Hippo signaling. We postulate that the interaction between SKI and TAZ in cardiac fibroblasts is arbitrated by LIMD1, an important intermediary in focal adhesion-associated signaling pathways. This study contributes to the understanding of the unique physiology of cardiac fibroblasts, and of the relationship between SKI expression and cell phenotype.

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Intermittent hypoxia mediated by TSP1 dependent on STAT3 induces cardiac fibroblast activation and cardiac fibrosis

eLife ◽

10.7554/elife.49923 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 5

Author(s):

Qiankun Bao ◽

Bangying Zhang ◽

Ya Suo ◽

Chen Liu ◽

Qian Yang ◽

...

Keyword(s):

Intermittent Hypoxia ◽

Cardiac Fibrosis ◽

Synergistic Effects ◽

Cardiac Fibroblasts ◽

Cardiac Fibroblast ◽

Ang Ii ◽

Fibroblast Activation ◽

Obstructive Sleep

Intermittent hypoxia (IH) is the predominant pathophysiological disturbance in obstructive sleep apnea (OSA), known to be independently associated with cardiovascular diseases. However, the effect of IH on cardiac fibrosis and molecular events involved in this process are unclear. Here, we tested IH in angiotensin II (Ang II)-induced cardiac fibrosis and signaling linked to fibroblast activation. IH triggered cardiac fibrosis and aggravated Ang II-induced cardiac dysfunction in mice. Plasma thrombospondin-1 (TSP1) content was upregulated in both IH-exposed mice and OSA patients. Moreover, both in vivo and in vitro results showed IH-induced cardiac fibroblast activation and increased TSP1 expression in cardiac fibroblasts. Mechanistically, phosphorylation of STAT3 at Tyr705 mediated the IH-induced TSP1 expression and fibroblast activation. Finally, STAT3 inhibitor S3I-201 or AAV9 carrying a periostin promoter driving the expression of shRNA targeting Stat3 significantly attenuated the synergistic effects of IH and Ang II on cardiac fibrosis in mice. This work suggests a potential therapeutic strategy for OSA-related fibrotic heart disease.

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scLink: Inferring Sparse Gene Co-expression Networks from Single-cell Expression Data

10.1101/2020.09.19.304956 ◽

2020 ◽

Author(s):

Wei Vivian Li ◽

Yanzeng Li

Keyword(s):

Gene Expression ◽

Single Cell ◽

Rapid Development ◽

Real Data ◽

System Level ◽

Expression Data ◽

Sequencing Technologies ◽

Gene Network Analysis ◽

Cell Gene Expression ◽

Cell Gene

AbstractA system-level understanding of the regulation and coordination mechanisms of gene expression is essential to understanding the complexity of biological processes in health and disease. With the rapid development of single-cell RNA sequencing technologies, it is now possible to investigate gene interactions in a cell-type-specific manner. Here we propose the scLink method, which uses statistical network modeling to understand the co-expression relationships among genes and to construct sparse gene co-expression networks from single-cell gene expression data. We use both simulation and real data studies to demonstrate the advantages of scLink and its ability to improve single-cell gene network analysis. The source code used in this article is available at https://github.com/Vivianstats/scLink.

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